Microbiol. Res.
(2001) 156, 293–301
http://www.urbanfischer.de/journals/microbiolres
Studies on composition and stability
of a large membered bacterial consortium degrading phenol
Saraswathy Ambujom
Regional Research Laboratory, Council of Scientific and Industrial Research, Trivandrum 695019, India
Present address: Department of Geology and Geochemistry, Stockholm University, 10691 Stockholm, Sweden,
Phone: +4686747728, Fax: +4686747855
Accepted: November 26, 2000
Abstract
A ten member microbial consortium (AS) consisting of eight
phenol-degrading and two non-phenol-degrading strains of
bacteria was developed and maintained in a fed-batch reactor
by feeding 500 mg l–1 phenol for four years at 28 ± 3°C. The
consortium could degrade 99% of 500 mg l–1 phenol after
24 hours incubation with a biomass increase of 2.6 × 107 to
4 × 1012 CFU ml–1. Characterization of the members revealed
that it consisted of 4 principal genera, Bacillus, Pseudomonas,
Rhodococcus, Streptomyces and an unidentified bacterium.
Phenol degradation by the mixed culture and Bacillus subtilis,
an isolate from the consortium was compared using a range of
phenol concentrations (400 to 700 mg l–1) and by mixing with
either 160 mg l–1 glucose or 50 mg l–1 of 2,4-dichlorophenol
in the medium. Simultaneous utilization of unrelated mixed
substrates (glucose/2,4-dichlorophenol) by the consortium
and Bacillus subtilis, indicated the diauxic growth pattern of
the organisms. A unique characteristic of the members of the
consortia was their ability to oxidize chloro aromatic com-
pounds via meta pathway and methyl aromatic compounds
via ortho cleavage pathway. The ability of a large membered
microbial consortia to maintain its stability with respect to its
composition and effectiveness in phenol degradation indi-
cated its suitability for bioremediation applications.
Key words: microbial consortium – phenol degradation –
composition – stability
Corresponding author: Saraswathy Ambujom
e-mail: ambujom@hotmail.com
0944-5013/01/156/04-293 $15.00/0
Introduction
For bioremediation applications, microbial consortia
are considered to have several advantages over a pure
cultures, such as greater stability and increased meta-
bolic capabilities. These characteristics enable the con-
sortium to overcome limitations for the complete bio-
degradation of toxis compounds (Davison et al. 1994).
This phenomenon has been exploited for the degrada-
tion of a large spectrum of environmental pollutants
including phenol (Khoury et al. 1992; Bisaillon et al.
1993; Gall and Winter 1993; Ambujom and Manilal
1995; Tawfiki Hajji et al. 1999), chlorinated phenols
(Puhakka et al. 1995) and nitrophenols (Hess et al.
1990; Silverstein et al. 1994).
The degradative efficacy of a microbial community
depends on the stability of the constituent members as
well as their ability to degrade or mineralize the target
compound. Nevertheless, in numerous degradative
studies, uncharacterized biological sludges/microbial
consortia have been used instead of defined or char-
acterized consortia. It is important from the perspective
of future applications to identify bacteria in the con-
sortium which are responsible for degradation as this
information could lead to optimization of degradative
processes as well as development of monitoring tools.
To date, however, there are only a few reports are
available on the nature of defined microbial consortia
capable of degrading toxic organic compounds. Davi-
son et al. (1994) reported on a nine member aerobic
bacterial consortium, consisting of morphologically and
physiologically different members of Pseudomonas,
Sphingomonas, and Alcaligenes species, capable of
degrading polychlorinated biphenyls. In another study,
Microbiol. Res. 156 (2001) 4 293
the presence of seven morphologically different mem-
bers in a methanogenic consortium increased the
ability to degrade pentachlorophenol (Juteau et al.
1995), phenol, ortho-, para- and meta cresol (Tawfiki
Hajii et al. 1999).
A consortium consisting of bacterial species with
varied physiology was shown to have a higher rate of
dicamba degradation than its individual members
(Fogarty and Tuovinen 1995). In addition, a three-
member consortium required morphologically different
members such a Pseudomonas fluorescens III,
Acinetobacter johnsonii and an oxidase positive spiral
shaped organism to degrade 347 mg l–1 of phenol in
20 hours of incubation (Khoury et al. 1992). Thus, the
above studies have indicated the importance of micro-
bial consortia and some of their constituent members
with respect to biodegradation of compounds.
However, reports on complex microbial communities
capable to degrading toxic organic compounds at ele-
vated concentration as well as in the presence of other
inhibitors is limited.
The objective of the present study was to identify the
different species in a large membered phenol degrading
consortia and to determine the role that each species
plays in the degradative process. The phenol removal
efficacy of the microbial consortium was compared
with reconstituted mixtures of bacterial isolates from
the consortium. The performance of consortium and its
best phenol degrading isolate was also evaluated under
elevated concentrations (400 to 700 mg l–1) as well as
in the presence of mixed substrates. In addition, the
stability of microbial consortium and its degradative
performance during extended periods of operation was
also investigated.
Materials and methods
Isolation. A phenol-degrading consortium was devel-
oped and maintained in a fed-batch reactor employing
the enrichment-culture technique that described earlier
(Ambujom and Manilal 1995). The reactor was fed
daily with enrichment media consisting of (g l–1)
K2HPO4, 0.5 ; MgSO4 · 7 H2O, 0.2 ; CaCl2 · 2H2O, 0.01;
NH4NO3, 3.0 ; FeSO4 · 7H2O, 0.01 ; and 1 ml of trace
elements solution (containing per liter: MnSO4 · H2O,
0.5 mg ; ZnSO4, 0.5 mg ; CuSO4 · 5 H2O, 5 µg and
CoCl2 · 6 H2O, 5 µg) and 500 mg l–1 phenol as the sole
carbon and energy source. The fed-batch reactor was
maintained for 4 years under the following conditions:
working volume 14 litre; stirring with air flow greater
than 4 Lpm; temperature, 28 ± 3 °C; dissolved oxygen,
1.4 mg l–1 ; pH 6–7; hydraulic retention time, 14 days;
dilution factor, 0.04h ; influent chemical oxygen
demand, 1487 mg l–1 ; effluent chemical oxygen
294 Microbiol. Res. 156 (2001) 4
demand, 297 mg l–1 and the biomass concentration in
the range of 2700–3000 mg l–1.
Microorganisms and growth conditions. Individual
phenol degrading bacterial strains of the microbial con-
sortium were isolated by plating on phenol agar
medium consisting of the enrichment media supple-
mented with 0.5 g l–1 of phenol and 1.5% agar. Non-
phenol degrading organisms were isolated by the spre-
ad plate method on nutrient agar medium. The Biolog
microbial identification system (Biolog Inc. Hayward,
CA. USA) was used to identify the isolates following
the procedure described by Klinger et al. (1992).
Inoculum preparation. Individual isolates from the con-
sortium were grown separately in 500 ml Erlenmeyer
flasks containing 100 ml enrichment media supple-
mented with 500 mg l–1 of phenol for phenol degraders.
For non-phenol degrading isolates, organisms were
cultured in Nutrient broth (100 ml). Cells were grown
for 72 hours, then separated and washed with phos-
phate saline buffer solution (NaCl, 8 g l–1 ; K2HPO4,
1.21 g l–1 ; KH2PO4, 0.3 g l–1, pH 7.3) by centrifugation
at 10,000 g for 15 minutes at 4 °C. The bacterial suspen-
sions (approximately 107 CFU/ml) were aseptically
transferred to experimental flasks for further studies.
Growth on derivatives of phenol and other aromatic
compounds as sole carbon and energy source by mem-
bers of the consortium were examined by inoculating
them in agar plates containing enrichment media
and 100 mg l–1 of 2-chlorophenol, 3-chlorophenol,
4-chlorophenol, 2,4-dichlorophenol, o-, m- and p-cre-
sol, nitrobenzene, 2-chlorobenzene, 4-hydroxy benzoic
acid, resorcinol, catechol or salicylate, respectively.
Identification of cleavage pathways for phenol degra-
dation. Production of the yellow product α-hydroxy
muconic semialdehyde (α HMS) or β-ketoadipate from
catechol was tested for detecting meta or ortho ring
fission pathways. (Shih et al. 1996). A 10 ml suspension
of phenol grown bacteria (72 hours) was concentrated
to 2 ml by centrifugation 10,000 g for 15 minutes at
4 °C. From the concentrate, 0.5 ml was re-suspended
in 2 ml of 0.2 M Tris buffer (pH 8.0) and 0.5 ml of
toluene was added to solublize the cell membrane and
the sample was shaken with 0.2 ml of 1.0 M catechol
solution. Appearance of the yellow colour within a
few minutes indicated meta cleavage activity. To 2.5 ml
of cell suspension, 1 g of (NH4)2SO4 was added and
incubated for 1 hour at 30 °C. The sample pH was
adjusted to ∼10 with 0.5 ml ammonia (5 N) and a
drop of 1% sodium nitroprusside was added to the
mixture. Appearance of a deep purple colour was ob-
served when ortho cleavage was active. Further, cate-
chol 2,3-dioxygenase indicative of meta cleavage was
assayed by the procedure of Nozaki (1970) and catechol
1,2-dioxygenase activity indicative of ortho cleavage
measured by the method of Dorn and Knackmuss
(1978).
Degradation experiments. The bacterial isolates were
inoculated separately or in combination based on their
physiological difference depending on whether they
followed the ortho pathway or meta pathway. Cultures
consisting of 2 members, 3 members, 4 members,
5 members, 6 members, 8 members, and 10 members
were inoculated into 500 ml Erlenmeyer flasks con-
taining 100 ml enrichment media added with 400, 450,
500, 550, 600, 650 or 700 mg l–1 phenol as sole carbon
source. To certain flasks prepared as above, was added
with 160 mg l–1 glucose/50 mg l–1 of 2,4-dichloro-
phenol to study the effect of unrelated dual substrates
on phenol degradation. Concentration of inoculum used Fig. 1. Phenol degraded by individual members of consor-
in each flask was made up to 0.6% (w/v) with all tium at 500 mg l–1 during 24 hours of incubation.
strains. Inoculated flasks were incubated on an orbital
shaker adjusted to 200 rpm at 28 ± 3 °C. Data presented
here in are the averages of triplicates.
Analytical methods. The concentration of phenol in
samples was analyzed spectrophotmetrically using the
4-aminoantipyrene method ASTM (1979). Viable
counts were determined by the serial dilution technique.
The biomass concentration used in each experiments
was determined as dry weight.

Results
Characteristics of microbial consortium (AS)
The microbial consortium (AS) enriched on 500 mg l–1
of phenol consisted of eight phenol degraders (nomina-
ted AS1 to AS8) and two non-phenol degraders (nomi-
nated AS9 and AS10). The composition of the microbial
consortium remained stable under a variety of incuba-
tion conditions and retained the ability to degrade
phenol. The identities of the bacterial isolates as deter-
mined using the Biolog system, are given in Table 1.
However, the gram positive strain (isolate AS8) could
not be identified using this method (Ambujom 1998).
Different trophic groups in the bacterial consortia were
evaluated by growing isolates of consortium in the pre-
sence or absence of phenol as the sole carbon source.
Testing of the mode of ring fission of aromatic com-
pounds showed that strains AS3, AS6 and AS7 induced
enzymes for an intra diol (ortho-cleavage) ring fission
to cleave aromatic nucleus, while other phenol de-
grading members of the consortium possessed enzymes
for an extra diol (meta-cleavage) ring fission. The non-
phenol degrading microorganisms of the consortium
did not possess enzyme activities for the fission of
benzene rings (Table 1).
Fig. 2. Phenol degraded by individual members of consor-
tium at varying initial concentration during 24 hours of incu-
bation.
Phenol degradation by individual members of the con-
sortium
Rate of phenol degradation was varied with each indi-
vidual member of the consortium in shaken flasks con-
taining 500 mg l–1 phenol as shown in Fig. 1. The
amount of phenol degraded by isolate AS5 (Bacillus
subtilis) was three times greater (360 mg l–1) than that
of isolate AS7 (Pseudomonas stutzeri). The degradative
performances of individual isolates were also varied
when assessed in samples containing range of phenol
feed ranging from 400 to 700 mg l–1. The strain AS5
showed an increase in degradation with an elevated
concentration of phenol up to 600 mg l–1, but most of
the other cultures failed to attain higher rate beyond
500 or 550 mg l–1 phenol (Fig. 2). Over a 24 hour

Microbiol. Res. 156 (2001) 4 295

Table 1. Morphological and biochemical characteristics of members of the consortium (AS).
Test AS1 AS2 AS3 AS4 AS5 AS6 AS7 AS8 AS9 AS10

Colony Discrete Rhizoid Circular Circular Rhizoid Rhizoid Irregular Circular Irregular Irregular
morphology
Size (µm) width 0.63 0.13 0.67 0.66 0.6 0.39 1 0.67 0.38 0.38
length 1.25 0.45 3.3 1.6 1.6 0.8 1.7 3.3 1.2 2.9
Shape Rod Rod Rod Rod Rod Rod Rod Rod Rod Rod
Gram reaction – + + – + + – – + +
Motility + + + + + – + – – +
Endospores – + + – + – – – + +
Catalase + + + + + + + + + +
Urease + – + + + – – – + –
Oxidase – + + + – + + + + +
Growth on Glucose + + + + + + + + + +
Sucrose – + + + + + + + + +
Fructose + + + + + + + + + +
Arabinose – + – + + + + + + –
Xylose + + – + + + + + + –
Mannose – + + + + + + + + +
Galactose – + + + + + + + + +
Rhamnose – + + + + + + + + +
Mannitol + + – + + + + + + –
i-Inositol – + + – + – – + – +
Hydrolysis of casein + + – + + – – – + +
Gelatin + – – – + + – + + –
Starch + – + – – + – – – –
Degradation of + – – – – + – – – –
tyrosine
Phenylalanine + + – – – – – – + –
Nitrate reduced to + – + + + + + + – +
nitrite
Indole formation + – – – + + + + – –
V. P test – – – – – – – – – –
H2S production + + + + + + + + + –
Growth at 0 °C – – – – – – – – – –
25 °C + + + + + + + + + +
37 °C + + + + + + + + + +
40 °C + + + + + + + + + +
50 °C – – + – + – – – – –
Cleavage Meta Meta Ortho Meta Meta Ortho Ortho Meta ND ND
pathway for
phenol degradation
Name of isolate Strepto- Bacillus B. co- Pseudo- B. Rhodo- P. ND B. B.
myces pumilus agulans monas subtilis coccus stutzeri sphaeri- cereus*
steonii putida rhodo- cus*
coccus
* Non-phenol degraders, ND: Not detected

growth period on phenol, strain AS5 had a faster growth
rate compared to other phenol degraders: microbial
numbers increased from an initial concentration of
5 × 107 to 3.9 × 109 CFU/ml (Table 2). Non-phenol
degrading members of the consortium (isolates AS9 and
AS10) were present in higher number (109 to 1012
CFU/ml) compared to phenol degrading members of
consortium.
Growth on aromatic compounds
Besides phenol, all phenol-degrading members of con-
sortium were able to utilize other aromatic compounds
as sole carbon and energy source (Table 3). However,
there were distinct differences between the strains with
respect to kind of aromatic compound utilized and the
velocity of growth. It is a unique adaptation for mem-

296 Microbiol. Res. 156 (2001) 4

Table 2. Profile of growth of bacterial isolates of the consor-
tium at 500 mg l–1 phenol during 24 hours of incubation.
Strain Initial concentration of Final concentration of
CFU/ml CFU/ml

AS1 4.6 × 107 5 × 107

AS2 3.8 × 107 4 × 108
AS3 2.6 × 107 3 × 107
AS4 3 × 107 3.6 × 108
AS5 5 × 107 3.8 × 109
AS6 4 × 107 4.3 × 108
AS7 3 × 107 3.5 × 107
AS8 2.8 × 107 5.6 × 107
AS9 1 × 109 2.5 × 1010
AS10 3.5 × 109 4 × 1012
Data are the mean average of triplicates; the average relative
standard deviation for all data points was 6% or less.

Fig. 3. Phenol degraded by various consortia reconstituted

with isolated members of microbial consortium (AS) at
500 mg l–1 of phenol.
bers of consortium to utilize chloro-aromatics by ortho-
cleavage as well as methyl-aromatics by ortho-cleavage
as indicated by catechol 2,3-dioxygenase and catechol
1,2-dioxygenase activity, respectively (data not shown).

Phenol degradation by reconstituted mixtures of bac-

terial isolates
Figure 3 shows the phenol degradation pattern of the
consortium (AS) and reconstituted mixtures of its iso-
lates during different period of incubation. Several
combination of bacterial consortia 1 member (AS5),
2 members (AS5 + AS8), 3 members (AS1 + AS2 + AS5),
4 members (AS1 + AS4 + AS5 + AS8), 5 members (AS1
Fig. 4. Profile of growth pattern of various reconstituted con-
+ AS2 + AS4 + AS5 + AS8), 6 members (AS1 + AS2
sortia from isolated members of AS at 500 mg l–1 phenol.
+ AS3 + AS4 + AS5 + AS7) 8 members (AS1 – AS8) and
10 members (AS1 – AS10) used in the present study
were enriched on the basis of their ability to degrade
phenol, with all combination included the best phenol
degrading member (AS5) of the community. About system could degrade smaller amounts of phenol (320
499.5 mg l–1 of 500 mg l–1 phenol was degraded by the and 266 mg l–1) over a 24 hour period when it was supp-
10 member consortium while the 2 member consortia lied at 400 mg l–1 or 700 mg l–1 phenol respectively.
could degrade only 385 mg l–1 during 24 hours of incu-
bation. Degradation activity increased gradually with
an increase in the number of members. The specific With mixed substrates
growth rate of members of the reconstituted mixtures When 500 mg l–1 of phenol was supplied in the presen-
also showed an increase from 0.013 to 0.065 l–1 (dry ce of 2,4-dichlorophenol or glucose, the microbial con-
weight) during 24 hours of incubation in the presence sortium could remove 475 and 485 mg l–1 of phenol
of 500 mg l–1 phenol (Fig. 4). during 12 hours of incubation (Fig. 6 a). Smaller
Results of the batch experiments conducted with amounts of phenol removal was observed when the
various reconstituted consortia at varying initial con- pure culture (AS5) was incubated in the presence of 2,4-
centration of phenol are presented in Fig. 5. The amount dichlorophenol or glucose: 45% of phenol was remo-
of phenol degraded by the consortium was 400, 499.5 ved in the presence of 2,4-dichlorophenol system while
and 630 mg at a phenol concentration of 400, 500 and 55% of phenol was removed in presence of glucose
700 mg l–1 respectively. In contrast, single member after 12 hours of incubation (Fig. 6 b). The addition of

Microbiol. Res. 156 (2001) 4 297

Table 3. Substrate spectra of members of consortium AS.
Compounds AS1 AS2 AS3 AS4 AS5 AS6 AS7 AS8 AS9 AS10

2-chlorophenol + + + + + + + + – –
3-chlorophenol + + + + + + + + – –
4-chlorophenol + + + + + + + + – –
2,4-dichlorophenol + + + + + + + + – –
p-cresol + + + + + + + + – –
m-cresol + + + + + + + + – –
o-cresol + + + + + + + + – –
Catechol + + + + + + + + – –
Salicylic acid + + + – + – + + – –
4-hydroxy benzoic acid + + + – + – + + – –
2-chlorobenzoic acid + + + – + – + + – –
Nitrobenzene + + + – + – + + – –
Resorcinol + + + – + + + + – –
Growth in agar containing 100 mg l–1 of respective compounds as sole carbon and energy source. + growth, – no growth.

Fig. 5. Phenol degraded by various reconstituted consortia

from isolated members of consortium AS at different initial
phenol concentration during 24 hours of incubation.

glucose to the system showed increased phenol degra-

dation by 4 and 9% for the consortium and the pure
culture (AS5), respectively.
It is interesting to note that even though the consor-
tium and pure culture follows the same growth pattern
(Fig. 7) to degrade phenol-glucose/phenol-2-4-di-
chlorophenol system, the rate of degradation by the
pure culture is less and similar to degradation of phenol
in an adapted concentration 500 mg l–1 of phenol
(Fig. 6 b). These results produce further evidence that
the pure culture has lesser degradation abilities compa-
red to that of consortium.
Figure 8 (a and b) shows the results of a 12 months
study of the biomass concentration, phenol degradation
and pH changes of bacterial consortium (AS). Phenol Fig. 6. Phenol degraded by consortium (a) and pure culture
degradation varied between 99.34 to 99.86% while pH (b) in presence of dual substrates during 24 hours of incuba-
remains neutral throughout the period of study. tion.

298 Microbiol. Res. 156 (2001) 4


Fig. 7. Profile of growth exhibited by consortium (a) and
pure culture (b) with or without dual substrates.
Fig. 8. Profile of Phenol degradation, pH (a) total bacterial
count of daily, weekly, and monthly variation (b) in microbial
consortium (AS) during 12 months study.
Discussion
In the present work, aerobic degradation of phenol was
studied with a large microbial consortium comprised of
10 organisms with varying abilities to degrade phenol.
Although phenol degradation by mircobial consortia
has been studied previously, the majority of studies
used uncharacterized mixed culture. (Khoury et al.
1992; Charest et al. 1999; Tawfiki Hajji et al. 1999). In
addition, none of the above studies investigated the
constitution and stability of a large membered bacterial
consortium involved in phenol degradation.
The stable bacterial consortia used in the present
study were enriched on the basis of their capacity to
degrade phenol: eight of ten organisms were phenol
degraders while renaining two organisms were non-
phenol degrading bacteria. It is interesting to note that
the non-phenol degraders were able to tolerate high
concentration of phenol (700 mg l–1) even though they
were unable to degrade the compound.
There were significant differences in the phenol
degradation rate by the isolates of consortium. The
most effective phenol-degrading member (AS5) of the
consortium was able to degrade 408 mg of 600 mg l–1
of phenol during 24 hours, while other members of con-
sortium failed to degrade half of the concentration
beyond 500 or 550 mg l–1 of phenol. This might be due
to accumulation of toxic intermediate compounds that
inhibit further phenol degradation. Further this results
indicated that each members of the consortium was
endowed with specific growth pattern and phenol
degradation ability.
Depending on the respective compound and the
organism, the degradation of mono-or di substituted
phenol by bacteria can be initialized via an oxidative
attack on the aromatic nucleus leading to catechols
which are then further oxidized to dihydroxylated sub-
strates for the following ring fission (Gibson and
Subramanian 1984). Usually bacteria utilizes extra
(meta) or intra diol (ortho) ring fission to cleave aroma-
tic nucleus, of these the former one is plasmid encoded
(Holloway and Morgan 1986) whereas the latter one is
chromosomally encoded one (Harayama et al. 1987).
Bacteria that degrade phenol via ortho pathways had a
slightly lower rate of phenol degradation compared to
meta cleaving members of consortium. However, none
of the member possessed both pathway for cleaving
phenol. Due to such differences, the possible inhibition
of phenol degradation may be reduced considerably.
Presumably, the consortium was able to perform stable
degradation because of its diverse biochemical path-
ways and degradation potential.
The results of substrate utilization tests on aromatic
compounds (Table 3), indicated that members of the
consortiun were capable of utilizing chloroaromatics by
Microbiol. Res. 156 (2001) 4 299
meta cleavage while methyl aromatics were cleaved via
ortho fission. These pathways were induced by catechol
2,3 dioxygenase and catechol 1,2 dioxygenase activity
respectively. The possession of both pathways plays an
important role in the degradative performance of the
microbial community as it prevents the inactivation of
catechol 2,3 dioxygenase activity by the accumulation
of acylchloride, a product of meta cleavage of 3-chloro-
catechol which can prevent the further oxidation of
catechols. A similar study by Mars et al. (1997) showed
Pseudomonas putida GJ31 possesed a meta cleavage
enzyme resistant to inactivation by the acylchloride.
Thus, organisms with an inducible pathway for degra-
dation of chloro/methyl phenols play an important role
in protecting the mircobial community against accumu-
lation of toxic intermediates during degradation.
Although the non-phenol degrading members of the
consortium were unable to utilize any of the aromatic
compounds as carbon sources, they were able to grow
on formate, a possible intermediate of aerobic phenol
degradation (Muller and Babel 1994). This result indi-
cated that non-phenol degraders did play an indirect
role, in phenol degradation by utilizing the metabolites
of degradation. However, this observation is contradic-
tory to that of Kramer and Kory (1992) who observed
less degradation of p-chlorophenol sample containing a
mixture of six bacterial isolates as compared to one of
its pure culture. A study of Coyle et al. (1993) that
showed 55% and 80% phenol removal by an
Pseudomonas putida F1 strain and an aerobic microbial
consortium at 420 mg l–1 of phenol in continuous cul-
ture system. However, the consortium and its best phe-
nol degrading isolate (AS5) used in the present study
was capable of degrading 99% and 72% of 500 mg l–1
phenol, respectively during 24 hours of incubation.
The presence of more than one organic compound in
the system may affect the biodegradative performances
of microorganisms (Zaidi and Mehta 1995). Many stu-
dies have reported the inhibitory or enhancing effects
of glucose or phenolic compound addition to phenol
degrading system (Rozich and Colvin 1986 ; Hess et al.
1993 ; Zaidi and Imam 1996). However, simultaneous
degradation of phenol and phenolic compounds by
mixed microbial consortia have only reported by few
workers (Suidan et al. 1983 a, b; Wang et al. 1988;
Charest et al. 1999 ; Tawafiki Hajji et al. 1999).
The present consortium and its isolates showed
simultaneous degradation of phenol and glucose
(Fig. 6 a and b). An enhancement of 16 – 20% phenol
removal was observed with pure culture and consor-
tium within 6 hours of incubation in presence of gluco-
se while total degradation was corresponding to that of
phenol alone system. Simultaneous degradation of
phenol and glucose by consortium and pure culture
indicated its diauxic nature of growth (Fig. 7) resulting
300 Microbiol. Res. 156 (2001) 4
from the metabolic control of intracellular flux balance
(Doshi and Venkatesh 1998). Similar enhanced de-
gradation of p-nitrophenol (1 mg l–1) by the addition of
20 mg l–1 of glucose was observed in a system inoc-
ulated with Pseudomonas sp. strain K (Zaidi and Imam
1996). While on the other hand, inhibition of phenol
degradation has been reported by the addition of gluco-
se to a heterogeneous population previously acclimated
to phenol (Rozich and Clovin 1986).
Addition of 50 mg l–1 of 2,4-dichlorophenol to be
consortium degrading 500 mg l–1 of phenol account-
ed the same degradation rate equivalent to that ob-
tained for 550 mg l–1 phenol by the end of incubation
(Fig. 6 a), although the degradation was faster initially.
Even though the consortium and pure culture follow
the same growth pattern to degrade phenol-glucose/
phenol-2,4-dichlorophenol system, the rate of degrada-
tion by the pure culture was less and similar to degra-
dation of phenol in adapted concentration 500 mg l–1
(Fig. 6 b). These results again showed that the pure
culture has less ability to degrade phenol compared to
the consortium.
Observation on daily, weekly and monthly variation
of bacterial count indicates that the present microbial
consortium was stable with respect to biodegradation of
phenol and constitutional aspects, without elimination
of a particular population, in spite of continuous shift in
the percentage composition of members in consortium
(Fig. 8). It works as a microcommunity capable of
mutually sustaining, constantly utilizing and dispensing
energy with less interdependency.
Obviously, there was increased phenol degradation
even under various concentrations and dual substrate
system by consortium which might be due to diverse
physiological and biochemical attributes from the
combination of more members. Hence, this work also
support the view that microorganisms constructed on
the basis of synergistic and commensalistic relation-
ships between the constituent members accomplished
degradative performance of a large consortium.
Stability of the system was maintained by combined
metabolic processes of individual members of the con-
sortium, enhanced by its biomass production, specific
growth rate and lack of accumulation of degradation
intermediates. Since there was no accumulation of toxic
metabolites in system, the present consortium offers
high potential for wastewater treatment facilities
treating high concentration of xenobiotics.
Acknowledgements
This work has been carried out as part of my doctoral pro-
gram. I thank Dr. Claire Adams, Dr. Albert Juhasz and
Dr. Farid Jan for a critical reading of the manuscript.
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